Culture of immortalized keratinocyte cell lines

The HaCaT cell line was originally purchased from the Cell Line Service (CLS, Eppelheim, Germany) as described elsewhere [13] and cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 2 mM L-glutamine–all obtained from Sigma (Sigma-Aldrich, Dorset, UK). The HaCaT-derivative keratinocyte line ‘HaCaTa’ was established as detailed previously [13] and cultured in keratinocyte serum free medium (KSFM) supplemented with epidermal growth factor and bovine pituitary extract (referred to as KSFM complete, KSFMc)–all obtained from Thermo Fisher Scientific (Loughborough, UK). All cells were routinely cultured at 37°C in a humidified atmosphere of 5% CO₂, whereas for cooling experiments the desired temperature was achieved using an LMS Series 1A Cooled Incubator with appropriate CO₂ provision for the duration of the incubation period (see below). Cells were passaged at ~80–90% confluence by removing media, rinsing with 0.1% EDTA in PBS (w/v) to aid disaggregation, and lifted using trypsin–EDTA solution (Sigma). For HaCaTa cells, the trypsin was inactivated by brief treatment with 5% FBS-containing KSFM medium, before routine culture in KSFMc. HaCaT cells were cultured in standard plasticware, whilst HaCaTa were cultured in Cell Plus (Cell+) plasticware (Sarstedt, Leicester, UK). HaCaT and HaCaTa cell lines were tested for Mycoplasma using the MycoProbe™ Mycoplasma detection assay (R&D Systems).